First Report of Rhizoctonia spp. Causing a Root Rot of the Invasive Rangeland Weed Lepidium draba in North America.

The exotic, invasive perennial rangeland weed Lepidium draba spreads rapidly and reduces native species diversity. The extensive root system of L. draba constitutes 76% of its biomass (4). Thus, searches have been done for biocontrol agents that target root tissue or that may interact with a weevil, Ceutorhynchus assimilis, that causes galls in the crown area of L. draba. An association of Rhizoctonia spp. with root tissue of plants galled by the weevil has been documented in Europe (3). The possible presence of soilborne pathogens similar to those found in the native range has been the subject of L. draba surveys in the United States. One such survey in 2008 detected a few plants with reddened and chlorotic foliage in a stand near Shepherd, MT. Such symptoms typically indicate the occurrence of soilborne diseases on L. draba in the native range of the weed (2). The site had shown a gradual increase in the range of detectable pathogens beginning with foliar pathogens in 1997. In 2010, at the Shepherd site, L. draba plants with similar (but more severe) symptoms to those seen in 2008 were noted in a different area of the stand. Excavation of the roots in both years revealed brown, sunken crown and root cankers. Pieces of root tissue were excised from the lesions and plated on acidified PDA and Ko and Hora medium. A non-sporulating fungus was isolated from three plants. Colonies of the isolates on PDA were typical of known Rhizoctonia spp. The 2010 isolates were determined to be multinucleate using DAPI and were paired with 14 tester (including subgroups) isolates of AG-1 to AG-4 on water agar. Anastomosis was observed between the multinucleate isolates and the AG-2-1 tester isolate. Sequence analysis of ITS of the rDNA of a multinucleate isolate (GenBank KJ545577) indicated 99% similarity with an accession of R. solani AG 2-1 (AB547381). The 2008 isolates were binucleate. A binucleate isolate, KJ545578, had 100% similarity with an isolate of Rhizoctonia spp. AG-A (AY927356). Pathogenicity tests consisted of planting 6-week-old seedlings of L. draba, one per pot, in ten 85-cm-diameter pots of pasteurized soil mix infested with Rhizoctonia-colonized barley grain that had been dried and milled. An inoculum level of ~8 CFU/g (1) of air-dried soil was established by most probable number calculations from fourfold dilutions of infested soil. Controls were the same number of plants in pasteurized potting mix. Results were recorded after 3 months in a greenhouse at 20-25°C. The test was repeated. Typically, R. solani caused mortality of six to eight plants, from which it was re-isolated, whereas binuclate isolates caused stunting and lower dry weight of L. draba. Control plants remained asymptomatic. This is the first report of R. solani and binucleate Rhizoctonia spp. on L. draba in North America. References: (1) A. J. Caesar et al. Plant Dis. 93:1350, 2009. (2) A. J. Caesar et al. Biol. Control 52:140, 2010. (3) A. J. Caesar et al. Plant Dis. 96:145, 2011. (4) R. F. Miller et al. Agronomy J. 86:487, 1994.

First Report of Spot Form Net Blotch Caused by Pyrenophora teres f. maculata on Barley in the Mon-Dak Area of the United States.

Pyrenophora teres Drechs. causes net blotch of barley, a common foliar disease in cultivation zones around the world. The disease occurs in two forms, namely a net form net blotch (NFNB) caused by P. teres f. teres and a spot form net blotch (SFNB) caused by P. teres f. maculata. As in other parts of the northern Great Plains, in the Mon-Dak area (western North Dakota and eastern Montana), NFNB is prevalent. SFNB was first reported in western Montana in 1983 (1) and more recently in eastern North Dakota in 2010 (3) but not in the Mon-Dak area. In the summer of 2011, unusual spot lesions that were surrounded by necrosis or chlorosis were observed on different barley cultivars in fields at Williston, ND, Nesson Valley, ND, and Sidney, MT areas. Diseased leaves from various barley cvs. from the three locations were transferred to water agar and incubated at room temperature for 24 h to induce sporulation. Morphological examination of conidia (45 to 169 × 15 to 21 µm) did not show significant differences from a known isolate of P. teres f. teres 0-1 (provided by Tim Friesen, ARS, Fargo, ND). For pathogenicity testing, six 14-day-old plants of barley cv. Tradition were sprayed until runoff with a 2,000 spore/ml suspension of two isolates from each location and the control P. teres f. maculata isolate DEN2.6 (provided by Tim Friesen). Plants were incubated first in a lighted humidity chamber for 24 h and then in a greenhouse for 7 days at 21°C. Regardless of inoculum source, spot lesions surrounded by necrosis or chlorosis, typical of SFNB, appeared on the inoculated leaves within 7 days. Fungi isolated from symptomatic leaves were identified as P. teres and the morphology of the conidia was undistinguishable from those of P. teres f. teres. All control plants which were sprayed with sterile distilled water were symptomless. The pathogenicity test was repeated. Rapid PCR detection and amplicon sequencing (2) of the internal transcribed spacer (ITS) region of ribosomal genes was performed on field and pathogenicity test leaf lesion samples to confirm the presence of P. teres f. maculata. DNA templates were prepared using the Extract-N-Amp Plant PCR Kits (Sigma Chemical Co., St. Louis, MO) and subjected to PCR using ITS1 and ITS4 primers. Amplicons were then purified and sequenced. The 585-bp nucleotide sequences of P. teres f. maculata from Mon-Dak area were submitted to GenBank under accession nos. PtmNES1 (JX187587), PtmSDY1 (JX187588), PtmSDY2 (JX187589), and PtmWIL1 (JX187590). The sequences from the four locations shared 100% similarity and also with P. teres f. maculata (EF452471) from GenBank while showing 10 nucleotide differences (99% similarity) with P. teres f. teres (EF452472).The results represent first report of SFNB in the Mon-Dak. Barley is one of the most important crops in the area. Resistance of the NFNB and SFNB of barley are controlled by different genes (4). Based on this report, SFNB therefore have to be considered in selection of barley cultivars for cultivation in the area. References: (1) H. E Bockelman et al. Plant Dis. 67:696, 1983. (2) R. T. Lartey et al. J. Sugar Beet Res. 40:1, 2003. (3) Z. H. Liu and T. L. Friesen. Plant Dis. 94:480, 2010. (4) O. M. Manninen et al. Genome. 46:1564, 2006.

First Report of Anthracnose Stem Canker of the Invasive Perennial Weed Lepidium draba Caused by Colletotrichum higginsianum in Europe.

Exotic perennial Lepidium draba, native to Eurasia, is an invasive weed in dense stands in rangelands and disturbed areas in several states of the western United States and an agricultural weed in the prairie provinces of Canada. To determine strategies, such as a potential multipathogen strategy (1), for biological control of the weed, surveys that included the native range were conducted in spring 2009 to detect diseases that occur on this weed. Several stunted and chlorotic plants were found scattered throughout a stand of L. draba growing in a vacant lot near Riddes, Switzerland (46°08'22.99″N, 7°9'19.02″E): ( http://maps.google.com/maps?source=earth&ll=46.13983490,7.15503250&layer= c&cbll=46.13983490,7.15503250&cbp=1,360,,0,5 ). Affected plants had reddish brown cankers on the lower stems, usually elongated and irregular in shape and slightly sunken. Insect injury was associated with the cankers. Symptoms often occurred on plants that were also infected with Rhizoctonia solani. After surface disinfestation with 0.1% sodium hypochlorite, tissue adjacent to and including lesions were plated on acidified potato dextrose agar and incubated at 20 to 25°C for 1 week. Zonate, dark gray colonies with sparse mycelia resulted that exhibited abundant, faintly pink spore masses with numerous dense clusters of black setae. Spores were single celled, hyaline, cylindrical to oval shaped, and 13.5 to 19.5 × 4 to 5.5 µm. Setae were 1- to 3-septate and 20 to 42 × 3 to 5 µm. These morphological traits correspond to Colletotrichum higginsianum. For pathogenicity tests, three 4-month-old L. draba plants were sprayed until runoff with a 106 conidia/ml suspension of the fungus and incubated for 72 h in plastic bags at 20 to 25°C in a quarantine greenhouse. Within 4 days, water-soaked lesions appeared that coalesced, resulting in chlorosis and collapse of inoculated leaves. Such symptoms are typical of infection by C. higginsianum and similar necrotrophic species (4). Fungi isolated from inoculated leaves were identified as C. higginsianum. To assess the host range of C. higginsianum, three plants each of turnip, radish, mustard greens, kale, broccoli raab, and Chinese cabbage, all in the Brassicaceae to which L. draba belongs, were inoculated with the same conditions used for the pathogenicity tests. Control plants in pathogenicity and host range tests were sprayed with sterile distilled water and all experiments were repeated at least once. All control plants were symptomless. Leaf necrosis occurred on radish and turnip and to a lesser extent on the lower leaves of Chinese cabbage and broccoli; numerous scattered dark necrotic flecks and small grayish leaf spots occurred on kale and mustard greens, respectively. These results are similar to previous studies (2,3) involving a cultivated species as the host in the field. The ITS1, 5.8S, and ITS2 sequences of this fungus (GenBank No. HM044877) were 99% similar to sequences of multiple isolates of C. higginsianum (GenBank Nos. AB042302, AB042303, AB455253, AJ558109, and AJ558110). To our knowledge, this is the first report of C. higginsianum on a wild species of the Brassicaceae, although a Colletotrichum sp. was reported on wild radish in Australia (1). References: (1) A. Maxwell and J. K. Scott. Australas. Plant Pathol. 37:523, 2008. (2) R. O'Connell et al. Mol. Plant-Microbe Interact. 17:272, 2004. (3) R. P. Scheffer. N. C. Agric. Exp. Stn. Tech. Bull. 1950. (4) H. Sun and J. Z. Zhang. Eur. J. Plant Pathol. 125:459, 2009.

Direct Polymerase Chain Reaction-Based Detection of Cercospora beticola in Field Soils.

Cercospora beticola, the causal agent of Cercospora leaf spot of sugar beet, survives as pseudostromata in infected sugar beet residues in the soil. Under optimal conditions, overwintering propagules germinate and produce conidia that are dispersed as primary inoculum to initiate infection in sugar beet. We developed a polymerase chain reaction (PCR) technique for rapid detection of C. beticola in field soils. Total DNA was first isolated from soil amended with C. beticola culture using the PowerSoil DNA Kit. The purified DNA was subjected to PCR in Extract-N-Amp PCR mix with CBACTIN primers over 35 cycles. The amplified products were resolved and compared by electrophoresis in 1% agarose gels. The PCR fragment size of C. beticola from the amended field soil correlated in size with the amplicon from control C. beticola culture DNA extract. Additionally, sample soils were collected from sugar beet fields near Sidney, MT and Foxholm, ND. Total DNA was extracted from the samples and subjected to PCR and resolved as previously described. The amplicons were purified from the gels and subjected to BigDye Terminator Cycle sequencing. All sequences from field soils samples, C. beticola-amended field soil, and pure culture were compared by alignment with a C. beticola actin gene sequence from GenBank. The result of the alignment confirmed the amplicons as products from C. beticola. Rapid screening for the presence of C. beticola in the soil by PCR will improve research capabilities in biological control, disease forecasting, and management of this very important sugar beet pathogen.

First Report of a Root and Crown Disease Caused by Rhizoctonia solani on Centaurea stoebe in Russia.

Spotted knapweed (SKW), Centaurea stoebe L., is a nonindigenous species that is invasive over large areas in the United States, especially in the west. It has been estimated that infestations of SKW cause $42 million in direct and indirect economic losses annually (2), and the weed could potentially invade 13.6 million ha of rangeland in Montana alone. Extensive efforts toward the control of SKW have included the release of 12 insects for biological control, four of which attack the crowns and roots of this short-lived perennial. To focus efforts to select potential soilborne pathogens, which could be applied in combination with insects, we conducted a survey for plant pathogens in the native range of SKW associated with damage caused by any root-attacking insects. Stunted and chlorotic SKW plants, which were colonized by larvae of Cyphocleonus spp., were found in June 1994 near the Novomar'evskaya Botanical Sanctuary (45°08'49.87″N, 41°51'02.05″E) in the Caucasus Region of Russia. A nonsporulating multinucleate fungus was isolated from the lower stem, crown, and upper root tissue of one such plant. Colonies growing on potato dextrose agar and Ko and Hora media were examined microscopically and identified as Rhizoctonia solani by the occurrence of robust, thick-walled, golden hyphae with right-angled branching and constrictions at the branch points. The anastomosis grouping of the one isolate was determined to be AG 2-2 IIIB after pairing it on water agar with 11 AG tester isolates representing all subgroups of AG 1 to AG 5. The hyphal diameter at the obvious point of anastomosis was reduced and cell death of adjacent cells was observed. In 2007, pathogenicity was determined by planting 12-week-old seedlings of SKW, one per pot, into 20 15-cm-diameter pots of a steamed greenhouse soil mix composed of sphagnum peat, sand, and Bozeman silt loam (1:1:1, vol/vol), pH 6.6, infested with R. solani-colonized barley grain that had been dried and milled. An inoculum level of 8 CFU/g of air-dried soil was determined by most probable number calculations from fourfold dilutions of infested soil. Controls were planted into noninfested soil. In both greenhouse tests, the isolate caused either mortality or a 93% mean fresh weight reduction of surviving plants, relative to the controls, after 8 months. R. solani was reisolated from necrotic root and crown tissue of dead and stunted plants but not from the controls. To our knowledge, this is the first report of R. solani occurring on SKW in Europe. The characterization and pathogenicity of Fusarium spp. isolated from insect-colonized roots of SKW in Europe was reported previously (1). References: (1) A. J. Caesar et al. BioControl 47:217. (2) S. A. Hirsch and J. A. Leitch, North Dakota Agricultural Economics Report No. 355. NDSU, Fargo. 1996.

Safflower: A New Host of Cercospora beticola.

Safflower is an oilseed crop adapted to the small-grain production areas of the western Great Plains, including the Northern Plains Area (NPA). In the NPA, safflower production is being evaluated for potential rotation with sugar beet. Safflower is susceptible to Cercospora carthami, whereas sugar beet is susceptible to C. beticola C. carthami has not been observed on safflower in the NPA but C. beticola is ubiquitous on sugar beet. Observation of unusual leaf spots on irrigated safflower cv. Centennial at Sidney, MT prompted this investigation of safflower as a potential alternate host of C. beticola. Safflower plants were inoculated with four isolates of C. beticola (C1, C2, Sid1, and Sid2) and incubated in growth chambers; leaf spot symptoms appeared between 3 and 4 weeks later. Polymerase chain reaction (PCR) amplification of extracts from lesion leaf tissue with C. beticola-specific primers produced fragments comparable with amplified fragments from purified cultures of control C. beticola. PCR assay of cultures of single spores from diseased safflower leaf lesions also produced fragments comparable with fragments from C. beticola cultures. Antibody that was raised from isolate C2 also bound to antigens from the single-spore cultures of the four C. beticola isolates. Inoculum from single-spore cultures from infected safflower also infected sugar beet and produced typical Cercospora leaf spot symptoms. Assay of these leaf lesions by PCR resulted in amplification of target fragments with the C. beticola-specific primers. Our results demonstrate that safflower is a new host of C. beticola.

Comparison between technetium-99m-teboroxime and thallium-201 dipyridamole planar myocardial perfusion imaging in detection of coronary artery disease.

Technetium-99m (TC-99m)-teboroxime is a new myocardial perfusion imaging agent. The purpose of this prospective study was to compare Tc-99m-teboroxime with thallium-201 imaging after the administration of dipyridamole. Thirty patients referred for the evaluation of chest pain were studied with both thallium-201 and Tc-99m-teboroxime dipyridamole scans (mean interval 2 days). Dipyridamole was administered at 0.142 mg/kg/min for 4 minutes. Planar imaging (3 standard views) was obtained at 5 and 240 minutes after the injection of 2.2 mCi of thallium-201. Tc-99m-teboroxime (18 to 25 mCi) was injected after dipyridamole infusion. A second injection, at rest, was repeated 4 hours later. Planar imaging (3 standard views of 1 minute/view for the first 2 views, and 90 seconds for the last view) was obtained 2 minutes after Tc-99m-teboroxime injection. Blinded reading was performed by 3 observers. Thallium-201 showed perfusion defects in 182 myocardial segments corresponding to 33 of 45 (73%) significantly stenosed coronary arteries (greater than or equal to 70% reduction in endoluminal diameter), and Tc-99m-teboroxime detected 160 abnormal segments corresponding to 29 of 45 (64%) stenosed arteries. Thallium-201 and Tc-99m-teboroxime studies were normal in 3 patients. In conclusion, this study shows that there is a good correlation in the imaging results found with thallium-201 and Tc-99m-teboroxime using dipyridamole infusion on both a segmental and a diagnostic comparison.

A monoclonal antibody to Candida albicans enhances mouse neutrophil candidacidal activity.

A monoclonal antibody (MAb) to Candida albicans (MAb B6.1) that protects against candidiasis and the nonprotective MAb B6 were compared for ability to support neutrophil (polymorphonuclear leukocyte [PMN]) candidacidal activity. Both MAbs are immunoglobulin M, and each recognizes distinct C. albicans mannan cell wall determinants. PMN candidacidal activity was assessed by transmission electron microscopy and by an in vitro killing assay. The results indicated that MAb B6.1, but not MAb B6, enhances ingestion and killing of yeast cells by PMN in the presence of serum complement.

Melanin production by a filamentous soil fungus in response to copper and localization of copper sulfide by sulfide-silver staining.

Gaeumannomyces graminis var. graminis, a filamentous soil ascomycete, exhibited enhanced cell wall melanin accumulation when exposed to as little as 0.01 mM CuSO(inf4) in minimal broth culture. Because its synthesis was inhibited by tricyclazole, the melanin produced in response to copper was dihydroxynaphthalene melanin. An additional hyphal cell wall layer was visualized by electron microscopy when hyphae were grown in the presence of copper and fixed by cryotechniques. This electron-dense layer was between the outer cell wall and the inner chitin layer and doubled the total wall thickness. In copper-grown cells that were also treated with tricyclazole, this electron-dense layer was absent. Atomic absorption spectroscopy demonstrated that up to 3.5 mg of Cu per g of fungal mycelium was adsorbed or taken up by hyphae grown in 0.06 mM CuSO(inf4). A method for silver enhancement was developed to determine the cellular location of CuS. CuS was present in cell walls and septa of copper-grown hyphae. Electron microscopy of silver-stained cells suggested that CuS was associated with the melanin layer of cell walls.

Melanin synthesis is associated with changes in hyphopodial turgor, permeability, and wall rigidity in gaeumannomyces graminis var. graminis.

Mycelia of Gaeumannomyces graminis var. graminis form large cells called hyphopodia with deeply lobed, melanized walls. Like appressoria produced by other pathogens, hyphopodia develop on hydrophobic surfaces, but it is not clear that hyphopodia function as platforms for host penetration. In appressoria, melanin synthesis is linked to the generation of enormous turgor pressures that provide the necessary force for plant penetration. In the present study, hyphopodial turgor was measured in a wild-type strain of G. graminis var. graminis, a mutant exhibiting constitutive synthesis of melanin (referred to as the dark mutant), and a melanin-deficient strain (thr). These experiments demonstrate that hyphopodia of the wild-type strain generate higher pressures than the dark mutant and that nonmelanized thr hyphopodia generate minuscule internal pressures. Melanization of the wall is also associated with an increase in its rigidity. These data correlate with differences in wall permeability consistent with a recent model for turgor generation by appressoria.